CN114366091B

CN114366091B - Microneedle patch for continuously monitoring or detecting in-vivo analytes, preparation method thereof and related device

Info

Publication number: CN114366091B
Application number: CN202210049073.4A
Authority: CN
Inventors: 金拓
Original assignee: Licheng Beijing International Biomedical Technology Co ltd
Current assignee: Licheng Beijing International Biomedical Technology Co ltd
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2023-08-22
Anticipated expiration: 2042-01-17
Also published as: WO2023134762A1; CN114366091A

Abstract

The invention relates to a microneedle patch capable of continuously monitoring or detecting in-vivo analytes such as blood sugar content without causing pain and skin injury, wherein the microneedle patch for detecting in-vivo analytes at least comprises a first transdermal microneedle standing on the patch, an electrochemical reaction tank is integrated in the first transdermal microneedle, the analytes react or act with detection components in the electrochemical reaction tank to form an electric signal, and the concentration level of the analytes is reflected by the magnitude of the detected electric signal; relates to a preparation method of a microneedle patch; also relates to a detection device comprising the microneedle patch that converts an electrical signal into a digital signal indicative of a concentration level of an analyte; the microneedle patch is wearable, and is painless, simple and economical in use.

Description

Microneedle patch for continuously monitoring or detecting in-vivo analytes, preparation method thereof and related device

Technical Field

The present disclosure relates to a microneedle patch capable of continuously monitoring or detecting in vivo an analyte such as blood glucose content without causing pain or skin damage, a method of manufacturing the microneedle patch, and a related detection device including the microneedle patch.

Background

Many biological information of the human body, such as blood sugar, blood fat, etc., need to be known through detection of blood or endocrine body fluid, and a substance capable of reacting to the biological information is referred to as an analyte in the present invention, such as the concentration of glucose in blood. To ensure the accuracy and precision of the detected analyte concentration, conventional analyte detection methods, which require invasion into the human skin, contact with blood or at least with endocrine body fluids, allow for the determination of the analyte and its content, are generally referred to as invasive or minimally invasive detection methods.

An invasive test method is exemplified by a blood glucose test strip disclosed in patent CN209979545U, which requires taking blood from a fingertip using a lancet on the test strip when detecting blood glucose, and then detecting the blood glucose concentration in the blood in combination with a detecting instrument. This type of testing device has been industrialized and the price of use is acceptable for most diabetics, and accurate data of blood glucose level is obtained by testing blood, but the invasive testing method similar to this solution has the following drawbacks: (1) The method can cause skin pain and skin injury, and is easy for a tester to generate contradiction emotion; (2) The testers can only intermittently measure the blood sugar level twice or three times a day, so that the situation that the blood sugar level is too high or too low cannot be found in time, the dosage of insulin or hypoglycemic agent cannot be adjusted according to the blood sugar change trend, and the possibility of occurrence of hypoglycemia is increased.

A minimally invasive detection method, such as chinese patent CN102472719B filed by large-scale headquarter-turn yazhi diabetes care company, claims priority from US patent 12495798, discloses an in vivo analyte monitoring device and method comprising a housing, a radio frequency receiver, an analyte sensor, a memory and a signal processor, wherein the components of the analyte sensor comprise a working electrode and a reference electrode, glucose oxidase coated on the working electrode, a semipermeable membrane for immobilizing the glucose oxidase on the working electrode, and the semipermeable membrane is made of a softer material than the analyte sensor, and it is necessary to implant the analyte sensor wholly or partially into the skin by means of an insertion device equipped with a rigid tip of 5-7mm, and remove the rigid tip from the skin by means of an ejection device of the insertion device. The analyte sensor is implanted into the skin for a long time, can continuously and accurately monitor the blood sugar concentration in the body of a user, solves the problem that the invasive detection method can only intermittently measure the blood sugar value, has higher detection accuracy, and realizes industrialization by a device for continuously monitoring the blood sugar based on the design principle, such as Abbott Free Style Libre series, but the too high use price can be accepted by fewer diabetics and cannot be popularized in the majority of diabetics. And a device for continuous monitoring of analyte levels based on this detection mechanism also suffers from the following drawbacks: (1) The electrochemical sensor can be implanted into the skin by means of the insertion device, the insertion device has a complex structure, and the electrochemical sensor is only used for one time and has high cost; (2) The semipermeable membrane cannot well coat the glucose oxidase, so that the glucose oxidase can gradually leak out along with the extension of the service time, and the detection signal is reduced; (3) Penetration of a rigid tip of 5-7mm into the skin can also give the user a foreign body sensation or discomfort; (4) The long placement of the electrochemical sensor in the subcutaneous tissue causes inflammatory and rejection reactions of the tissue, while the high disposable costs require the device to remain subcutaneous for as long as possible.

The devices for continuously monitoring the analyte level disclosed in the patent EP3524150B1, EP3641636B1, US11166657B2, US10932709B2 and US10932699B2 and commercialized by large medical instruments such as meiton force medical instruments, microtai medical instruments and kelite medical instruments are designed based on the minimally invasive detection principle, but commercial products cannot be popularized in the majority of diabetics due to the problems of excessively high use price and use comfort.

To avoid pain and injury, the scientific community has conducted studies on noninvasive detection and monitoring methods, and the methods for noninvasive detection and monitoring under investigation are roughly classified into two categories: (1) Irradiating with a non-invasive light source capable of penetrating human tissue and tissue fluid, such as near infrared, and estimating the concentration of the target component from diffuse reflection or Raman spectrum of the light source; (2) Exocrine body fluids such as tears, saliva, sweat, urine, etc. are collected and tested. The above method has many limitations, such as near infrared is the frequency of penetrating tissues and body fluids in various light sources, and has no molecular motion (rotation, vibration, energy level transition, etc.) corresponding to the near infrared, and diffuse reflection is difficult to carry chemical information. The raman effect of near infrared falls into the molecular vibration level, the molecular vibration mode with raman activity is not much, and the vibration structure of the biological molecule is similar, so that the raman spectrum of the analyte is difficult to distinguish from the mixed spectrum under the condition that the in-vivo in-situ purification of the analyte cannot be carried out, even though fourier transformation is carried out. Some research teams set aside the reliance on specific chemical mechanisms, attempting to monitor blood glucose levels and their changes by near infrared diffuse reflectance through big data alignment. However, the human body has too many nonspecific factors affecting near infrared diffuse reflection, so far the scientific community has not obtained accurate and regular results, and can not continuously obtain accurate blood glucose concentration data through a near infrared irradiation method.

The latter method also does not allow accurate measurement of the concentration levels of analytes because of the tissue membrane separation between the exocrine fluid used and the blood and endocrine fluids, the concentration of the target component being orders of magnitude different from that of the endocrine system. In other words, such a large concentration difference causes an absolute error in measurement with a relative error of order of magnitude magnification. Such differences make it difficult to provide accurate references for the drug treatment of some diseases with measurement accuracy; for example, it is difficult to give accurate insulin doses for glycemic control.

How to continuously obtain accurate detection of blood sugar or other in-vivo analytes without touching the person to be tested due to the pain of needling, and brings new challenges to the in-vivo analyte index monitoring field. The microneedle does not cause a tingling sensation when penetrating the skin, and the scientific community is beginning to pay attention to the research of analyte detection devices based on the microneedle. Existing microneedle-based analyte detection devices fall into two categories in terms of detection means: the first is to form micropores on the skin by utilizing the puncture property of the microneedle, and then draw out the internal body fluid through the micropores for detection; the second type is to assemble microneedles into biosensor inserts to detect the concentration levels of analytes.

Patent CN109199400B discloses a blood glucose electrochemical sensor based on a microneedle array, which is designed based on the first detection mode, and comprises a microneedle array electrode, a reference electrode one, a reference electrode two and a conductive electrochemical detection area storing biological recognition molecules. When in use, the microneedle array electrode pierces the skin, then rebounds and exits the skin, and body fluid is guided onto the conductive elastic hydrogel storing biological recognition molecules in the conductive electrochemical detection area through micropores formed on the skin by the microneedle array, so as to detect blood sugar. The method needs to lead out the body fluid for re-measurement, and has long physical delay, so that the real-time detection performance is reduced; the condition of doping sweat and other interference components can occur in the body fluid exudation process, so that the detection accuracy is reduced; and the skin at the position of body fluid extravasation is easy to cause eczema, and the problems of inflammation and the like are caused. The diabetes patients are easy to have high blood sugar after meal or the insulin secretion disorder patients need to monitor blood sugar immediately in order to cope with the unstable blood sugar caused by the insulin secretion disorder, and the method is not suitable for instant monitoring because of the monitoring delay caused by the need of leading out body fluid, and is not suitable for continuous monitoring or high-frequency detection of the in-vivo biological substance level, and the monitoring or high-frequency detection is necessary for accurate drug treatment; such as blood glucose level-physical state-correlation of insulin dosage and rate.

How to perform the monitoring or detection of the analytes in a noninvasive, painless, immediate and continuous manner and have better affinity with human bodies when performing the monitoring or detection becomes a new challenge of scientific research.

Disclosure of Invention

The inventors have studied the monitoring or detection technique of in vivo analytes based on the current situation of monitoring or detection of in vivo analytes, and have found that to achieve a non-invasive, painless effect in the monitoring or detection process requires that the detection tool be brought into contact with nerve endings or not after penetrating the dermis layer as little as possible. Since nerve endings in the dermis layer are mainly distributed in the dermis layer deep away from the epidermis layer, the inventors believe that the length of penetration of the skin by the detection tool is short enough to allow contact with body fluid in the dermis layer shallow near the epidermis layer without touching the nerve endings and causing pain. The inventor considers that the damage to the skin caused by the detection tool penetrating the skin is about to fall into the reversible damage range, and when the detection tool is separated from the skin, the skin damage caused by the detection tool can be recovered in a short time, so that the noninvasive effect can be achieved. The inventors believe that a microneedle of smaller length and diameter in this case can achieve a non-invasive, painless effect when used as a detection tool to penetrate the skin. However, most of the conventional microneedles are made of metal, and have poor affinity for skin although they can penetrate the skin, and cannot effectively fix the detection component therein, so that there is a serious rejection reaction between the microneedles made of metal and the skin. The inventors considered that the microneedle is made of a polymer material having a good biocompatibility, but the polymer material needs to be sufficiently hard to penetrate the skin in a dry state and to be capable of containing or embedding a detection component, and the analyte in the body fluid should be capable of contacting with the detection component immobilized in the polymer material. In view of the above, the inventors have found that a polymer material needs to have a network structure, and a detection component is fixed in a mesh to avoid leakage of the detection component, and also that an analyte in a body fluid penetrates into the network structure to contact with the detection component, and that the polymer material needs to have a certain hardness in a dry state to be able to penetrate into the skin.

Polyvinyl alcohol (PVA) is known to those skilled in the art of polymer synthesis to have a good biocompatibility, but it is required to form a gel of a network structure of polyvinyl alcohol to achieve immobilization of a detection component and diffusion of an analyte into the inside thereof, and to dissolve in body fluid. The inventors have unexpectedly found that polyvinyl alcohol subjected to freeze-thawing treatment can form a network structure that swells without dissolution after absorbing water and has hardness to penetrate the skin in a dry state, and have developed a microneedle patch for detecting blood glucose based on the above ideas.

The first aspect of the invention relates to a microneedle patch for continuously monitoring or detecting an analyte in a body, comprising at least one first transdermal microneedle standing on a skin-contacting surface of a patch substrate layer, the microneedle having a sharp shape at an end remote from the patch substrate layer; the first transdermal microneedle is integrated with an electrochemical reaction cell, and an analyte electrochemically reacts or acts with a detection component contained in a matrix of the first transdermal microneedle in the electrochemical reaction cell to form an electric signal, and the concentration level of the analyte is reflected by the magnitude of the detected electric signal.

Preferably, the matrix of the first transdermal microneedle has a network that swells after water absorption without dissolution, allowing the analyte to diffuse into or migrate away from the matrix of the first transdermal microneedle, while preventing the detection component therein from migrating away.

Preferably, the patch further comprises at least one second transdermal microneedle which is vertical to the skin-contacting surface of the patch substrate layer, wherein the second transdermal microneedle does not contain detection components, the electrochemical reaction tank in the first transdermal microneedle comprises a working electrode, the second transdermal microneedle comprises a reference electrode, and the reference electrode in the second transdermal microneedle and the working electrode in the first transdermal microneedle are connected into a measuring loop.

Preferably, the electrical signal generated in the measurement loop comprises an electrical value in quantitative relation to chemical equilibrium and an electrical value in quantitative relation to electrochemical reaction rate.

Preferably, the first transdermal microneedle has a length in the range of 600-1500 μm and a diameter at a maximum cross-sectional area in the range of 200-700 μm.

Preferably, one end of the working electrode in the electrochemical reaction tank is inserted into the first transdermal microneedle matrix, and the other end extends out of the back surface of the skin-contacting surface of the patch substrate layer to form a first electric contact.

Preferably, one end of the reference electrode is inserted into the second transdermal microneedle matrix, and the other end extends out of the back surface of the skin-facing surface of the patch substrate layer to form a second electric contact.

Preferably, the working electrode in the electrochemical reaction cell comprises at least an inert conductor embedded in the first transdermal microneedle matrix, wherein the inert conductor is selected from one or a combination of several of platinum, gold, copper, silver and carbon materials.

Preferably, the working electrode further comprises a terminal post electrically connected to the inert conductor and transmitting an electrical signal formed on the inert conductor to the outside of the first transdermal microneedle.

Preferably, the first transdermal microneedle matrix in the first transdermal microneedle is prepared from a hydrophilic polymer.

Preferably, the preparation raw materials of the first transdermal microneedle matrix at least comprise polyvinyl alcohol as a main material.

Preferably, the polyvinyl alcohol used as the preparation raw material of the first transdermal microneedle substrate is completely-alcoholyzed polyvinyl alcohol and/or a mixture of completely-alcoholyzed polyvinyl alcohol and partially-alcoholyzed polyvinyl alcohol.

Preferably, the preparation raw materials of the first transdermal microneedle substrate can further comprise auxiliary materials, wherein the auxiliary materials are selected from one or a mixture of more of dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate, sodium carboxymethylcellulose and polyethylene glycol.

A second aspect of the invention relates to a device for processing electrical signals generated by a microneedle patch for continuous monitoring or detection of an analyte in a body, comprising at least a signal processor electrically connected to a measurement circuit formed by a microneedle patch for continuous monitoring or detection of an analyte as described above, monitoring the electrical signals in the measurement circuit and converting the electrical signals into digital signals related to the level of the analyte.

A third aspect of the present invention is directed to a device for continuously monitoring or detecting an analyte in a body, comprising at least the microneedle patch described above, optionally having an adhesive layer adhered to the skin; and

and a signal processor electrically connected to the measurement circuit between the first and second transdermal microneedles, monitoring the electrical signal in the measurement circuit and converting the electrical signal to a digital signal related to the analyte level.

Preferably, the device for continuously detecting the in-vivo analyte further comprises a connecting plate, plug-in elements are arranged on two sides of the connecting plate, the microneedle patch is detachably connected with the connecting plate through the plug-in elements on one side of the connecting plate, the signal processor is detachably connected with the connecting plate through the plug-in elements on the other side of the connecting plate, and a measuring loop formed by the signal processor and the microneedle patch is electrically connected through the connecting plate.

The device for continuously monitoring or detecting an analyte in a body according to the present invention is a wearable device.

A fourth aspect of the invention relates to a method for preparing a microneedle patch for continuous monitoring or detection of an analyte in a body, comprising the steps of:

step 1: casting the molding preparation liquid of the microneedle substrate into a casting mold with a sharp end, which is placed on a running suction device, and then continuously casting the molding preparation liquid of the microneedle patch substrate layer into the casting mold, wherein the casting mold is placed in the running suction device;

Step 2: freezing and thawing the molding preparation liquid in a casting mold;

step 3: inserting an electrode into the casting mould, and extending the tail end of the electrode out of the surface of the molding preparation liquid;

step 4: repeating the step 2 for a plurality of times; and

step 5: the microneedle patch preparation was dried at room temperature to shrink and then dried to complete cure.

Preferably, the casting mold used for the method for preparing a microneedle patch for continuously monitoring or detecting an analyte in a body has a microneedle hole for microneedle molding, and the molding preparation liquid of the microneedle substrate is sucked into the microneedle hole by a suction means.

The beneficial effects are that:

(1) According to the technical scheme, the electrochemical reaction tank is concentrated in the first transdermal microneedle, the substrate of the first transdermal microneedle can penetrate into the epidermis to reach the dermis layer, the electrochemical reaction tank is brought into the skin when the first transdermal microneedle penetrates into the skin, a complex disposable insertion device or an application device is not needed to implant the electrochemical reaction tank into a human body, the structure of an analyte detection device is simplified, the use cost is saved, the instant measurement is realized, the low price requirement of a common patient for continuously monitoring blood sugar is met, the inconvenience that the patient is attached for a long time due to the cost is solved, and the contradiction emotion of the patient to be tested in the test is lightened.

(2) The first transdermal microneedle substrate in the technical scheme not only can bring the electrochemical reaction tank into the skin, but also allows the analyte to diffuse into or move away, prevents the detection component from moving away, can better fix the detection component therein, can not leak the detection component in the long-term monitoring process, and can ensure the long-term accurate monitoring of the analyte.

(3) The first transdermal microneedle matrix is frozen and thawed to form a reticular structure, so that the activity of detection components and enzyme is not reduced or influenced, the peristaltic motion of the enzyme during catalysis is not hindered, the enzyme activity can be well maintained, the detection precision of the microneedle patch is improved, the practicability and the continuous measurement precision of the microneedle patch are ensured, the microneedle patch for continuously monitoring or detecting in-vivo analytes has an industrialization prospect, and the microneedle patch is widely accepted and applicable by diabetics.

(4) The high polymer material used by the microneedle patch is an affinity material, so that the microneedle patch has little uncomfortable feeling even if worn for a long time, and the matching degree of the microneedle patch for long-term wearing by a user according to the requirements of doctors is improved.

The beneficial technical effects brought by the invention are not obvious from the effects, and more details are shown in the detailed description and the examples.

Drawings

The drawings illustrate only some embodiments and therefore should not be considered limiting of scope.

1-first transdermal microneedle, 12-groove, 2-platinum wire, 3-copper bar, 4-patch substrate layer, 5-nanoampere meter, 6-second transdermal microneedle, 7-microneedle patch substrate layer and 8-first electric contact.

Fig. 1: schematic of a measurement loop formed by the first transdermal microneedle and the second transdermal microneedle.

Fig. 2: when the detection component is glucose oxidase, an electrochemical reaction in the first transdermal microneedle is schematically represented.

Fig. 3: the microneedle patch measures the current profile over time after the loop is turned on.

Fig. 4: a graph of constant reaction current versus glucose concentration for microneedle patches carrying different glucose oxidase enzymes.

Fig. 5: a linear plot of constant reaction current versus glucose concentration for a microneedle patch carrying 10wt% glucose oxidase was fitted.

Fig. 6: constant response current value versus glucose solution graph for microneedle patches used for different days.

Fig. 7: at different glucose addition levels, the microneedle patch was compared to glucose measurements and glucose true values for the bid Abbott Free Style Libre 3 (attire millimeter needle patch).

Fig. 8: the enrichment value on the working electrode versus the circuit off time.

Fig. 9: a graph of saturation electrical value versus glucose concentration for microneedle patches carrying different glucose oxidase enzymes.

Fig. 10: saturated capacity versus off time for different glucose concentrations.

Fig. 11: at different temperatures, a graph of saturated electric quantity value-glucose solution concentration relationship is enriched.

Fig. 12: a plot of microneedle patch saturation electrical value versus glucose concentration for 10wt% glucose oxidase loading.

Fig. 13: a linear fit plot of microneedle patch saturation electrical value versus glucose concentration for 10wt% glucose oxidase loading.

Fig. 14: graph of saturation electric quantity value enriched on working electrode on different days versus glucose concentration

Fig. 15: microneedle patch with adhesive film.

Fig. 16: current versus time plot for microneedle patch test rabbits for blood glucose values.

Fig. 17: rasch blood glucose meter is used for testing the relationship graph of the blood glucose value of rabbits with time when different glucose injection amounts are measured.

Fig. 18: microneedle patch versus time graph of blood glucose level versus time for rabbits tested with a rogowski blood glucose meter.

Fig. 19: the microneedle patch and the rogowski blood glucose meter were tested for a comparison of rabbit blood glucose value versus time for 3 consecutive days.

Detailed Description

The invention describes a microneedle patch for continuously monitoring or detecting an analyte in a body, comprising at least one first transdermal microneedle standing on a skin-contacting surface of a patch substrate layer, the microneedle having a sharp shape at an end remote from the patch substrate layer; the first transdermal microneedle is integrated with an electrochemical reaction cell, and an analyte electrochemically reacts or acts with a detection component contained in a matrix of the first transdermal microneedle in the electrochemical reaction cell to form an electric signal, and the concentration level of the analyte is reflected by the magnitude of the detected electric signal.

The first transdermal microneedle of the present invention has an electrochemical reaction cell integrated therein, and the electrochemical reaction cell of the present invention has an electrochemical reaction element, and as an example of an electrochemical reaction cell, the electrochemical reaction cell includes a working electrode that participates in a reaction with an analyte, and an enzyme or other active ingredient that catalyzes the reaction, and the enzyme or other active ingredient is referred to as a detection ingredient in the present technical scheme. When the in-vivo analyte is required to be detected, the first transdermal microneedle can be directly inserted into body fluid of the skin, an electric signal is formed in the electrochemical reaction tank by the in-vivo analyte, the concentration level of the analyte is judged according to the quantitative relation between the electric signal and the concentration of the analyte, the first transdermal microneedle can be directly inserted into the electrochemical reaction tank into the skin without an insertion device with a complex structure, the complexity of the monitoring device is simplified, and the production cost of the monitoring device is reduced. The first transdermal microneedle comprises a first transdermal microneedle matrix. The micro-needle shallow insertion type attaching process has no pain, the interference emotion of a patient is reduced, a sharp guide used by the blood sugar minimally invasive monitoring attaching device in the market and a relatively expensive disposable catapulting attaching device are not needed, the duration of one-time attaching is not limited by cost, the micro-needle patch can be optimized along with the living habit of a user, and the low use cost enables the micro-needle patch for continuously monitoring or detecting in-vivo analytes to be widely used in a large number of diabetics.

As a preferred technical solution, the shape of the microneedle having a sharp shape at the end far from the patch substrate layer may include, but is not limited to, one or a combination of a conical shape, a biconic shape, and a pyramid shape, and the biconic shape may be a biconic combination having one tip or a conical and pyramid combination having one tip.

As a preferred embodiment, the matrix of the first transdermal microneedle has a network that swells after absorption of water without dissolution, allowing the analyte to diffuse into or migrate out of the matrix of the first transdermal microneedle while preventing the detection component therein from migrating out.

The first transdermal microneedle matrix is inserted into skin to penetrate epidermis layer of skin to reach dermis layer, and absorbs body fluid in dermis layer to form swelling and insoluble network structure, and the first transdermal microneedle matrix has network structure to realize fixation of detection component and free diffusion of analyte into and out of the dermis layer. Analyte diffusion into the first transdermal microneedle matrix produces a detectable electrical signal in the electrochemical reaction cell, and analyte concentration is determined based on a quantitative relationship between the electrical signal and the analyte. The first transdermal microneedle substrate allows the analyte to diffuse into or move away from the first transdermal microneedle substrate, and prevents the detection component from moving away from the first transdermal microneedle substrate, so that the analyte and the detection component can be effectively contacted when the first transdermal microneedle is in the analyte in-vivo environment, the detection component can be ensured to exist in the first transdermal microneedle stably for a long time, the service time of an electrochemical reaction cell is prolonged, and the continuous monitoring is ensured.

The second transdermal microneedle comprises a second transdermal microneedle matrix and a reference electrode positioned in the second transdermal microneedle matrix. The structure, material, and the like of the second transdermal microneedle substrate are the same as or similar to the structure, material, and the like of the first transdermal microneedle substrate. The detection component reacts with the analyte in the body fluid to produce an electrical signal that can be detected, such as electrons or positive charges or an electrical quantity enriched on the working electrode; or the electrons or the electric quantity enriched on the working electrode of the electrochemical reaction cell by the interaction of the analyte with the detection component in the electrochemical reaction cell and the working electrode are converted into electric signals which can be detected, such as current, electric quantity or electromotive force, by forming a loop with the reference electrode.

The number of the first transdermal microneedles in the electrochemical reaction measurement loop can be 2, 3, 4 or more, the number of the second transdermal microneedles can be 2, 3, 4 or more, the arrangement of the first transdermal microneedles can not only increase the strength of an electric signal, but also increase the stability of a microneedle patch, and the arrangement of the second transdermal microneedles can improve the stability of the electric signal. In the technical scheme, two first transdermal microneedles and one second transdermal microneedle are taken as examples for explanation, and three microneedles can form a stable structure.

Taking the example of the measurement loop formed by electrically connecting two first transdermal microneedles and one second transdermal microneedle in fig. 1, the detection mechanism of the microneedle patch will be described. As shown in FIG. 2, the electrochemical reaction and the generation of an electric signal are described by taking a case where the detecting component is glucose oxidase as an example. The first transdermal microneedle with working electrode and immobilized glucose oxidase and the second transdermal microneedle with reference electrode are passed through the epidermis layer of the skin and into the body fluid in the dermis layer. At this time, the first transdermal microneedle and the second transdermal microneedle absorb moisture in the body fluid to swell, glucose in the body fluid diffuses into the first transdermal microneedle to undergo electrochemical reaction as shown in fig. 2, and glucose is adsorbed and reacted on a catalytic site of glucose oxidase to generate gluconolactone in the electrochemical reaction, and then is hydrolyzed into gluconic acid; the catalytic site of the glucose oxidase is converted into a reduced state, and the glucose oxidase in the reduced state is oxidized into an oxidized state with catalytic activity under the action of oxygen in body fluid, and simultaneously one molecule of hydrogen peroxide is released; the hydrogen peroxide loses two electrons on the surface of the working electrode, generating a molecule of oxygen and two hydrogen ions. Oxygen participates in the conversion of glucose oxidase, hydrogen ions become a component of gluconic acid, and the product and reactants realize local circulation. Electrons are absorbed by the working electrode in the first transdermal microneedle, electrochemical reaction occurs on the working electrode, electrochemical reaction does not occur on the reference electrode, the working environment of the working electrode is different from that of the reference electrode, the electrode potentials are different, potential differences are generated between the working electrode and the reference electrode, current is generated between the working electrode and the reference electrode after the working electrode and the reference electrode are electrically connected to form a loop, and the concentration level of the analyte is estimated through the quantitative relation between the current and the analyte. As shown in fig. 2, in the electrochemical reaction, glucose on the left side of the reaction formula is oxidized to lose electrons, and electrons are obtained from the working electrode on the right side, whereas the oxidation state and the reduction state of glucose oxidase, and hydrogen peroxide and oxygen circulate inside the reaction system, so that the supply of oxygen does not become a factor affecting the reaction rate.

As a preferable embodiment, the detection component is selected from one or more of enzyme, hormone, antibody, DNA, RNA, and modified enzyme.

Enzymes that may be cited include, but are not limited to, one or more of glucose oxidase, catalase, amylase, creatine kinase, lactate oxidase, cholesterol oxidase.

Hormones that may be mentioned include, but are not limited to, growth hormone and/or thyroid stimulating hormone.

The enzyme is a nanogold-modified glucose oxidase.

The modification method of the nano-gold modified glucose oxidase in the scheme can be as follows: and finally, the glucose oxidase with the coenzyme removed is combined with the composition of the coenzyme-nano gold particles again, and the active center of the modified glucose oxidase is directly connected with the nano gold and then connected with the working electrode, so that the electron transfer efficiency is improved. When glucose is catalyzed by the nano-gold modified glucose oxidase, glucose is oxidized into glucolactone, the glucose is changed into a reduced state, the reduced state nano-gold modified glucose oxidase is oxidized on the working electrode to be changed into an oxidized state again, two molecules of hydrogen ions and two electrons are formed, and the generated electrons are detected by the working electrode to form corresponding electric signals.

As a preferable technical scheme, the detection component is glucose oxidase.

As a preferred embodiment, the electrical signal generated in the measuring circuit comprises an electrical value in quantitative relation to the chemical equilibrium and an electrical value in quantitative relation to the electrochemical reaction rate. When the endocrine body fluid diffuses into the first transdermal microneedle at a glucose concentration far below the catalytic site of glucose oxidase, the reaction rate is independent of the glucose oxidase loading and has a primary relationship to the glucose concentration. The overall reaction rate, i.e. the rate of electron generation, is then also a linear dependence between the magnitude of the current and the glucose concentration, which becomes a constant current when the current stabilizes. Thus, the concentration of glucose in the body fluid is known by the constant current generated between the working electrode and the reference electrode. The glucose concentration in the body fluid is similar to that in blood, and thus the blood glucose concentration can be known. The above detection mechanism and the following measurement modes of electric signals are equally applicable to the rest of detection components, and when the detection component is glucose oxidase, the following measurement modes of two electric signals are specifically described:

(1) When the constant current signal in the electrochemical reaction is strong and stable measurement can be performed, the constant current in the continuous on-circuit can be measured to reflect the glucose concentration in the body fluid, and then reflect the blood glucose value. Under the condition that the measuring loop is continuously connected, the current in the measuring loop can be continuously recorded, read and subjected to data processing, so that the blood glucose value can be monitored in real time. From the administration perspective, the collection of blood glucose data can also be intermittently performed, for example, the interval time can be controlled to be 5-10 minutes, continuous monitoring of blood glucose is realized by continuously and intermittently reflecting the trend of blood glucose change of the blood glucose, and the collection of intermittent data can reduce the storage requirement of a data storage. The glucose concentration may be obtained under a known constant current condition by an empirical formula between the constant current and the glucose concentration.

(2) When the current signal in the electrochemical reaction is weak enough to maintain stable measurement, the amount of electricity enriched on the working electrode in the open circuit can be measured instead of the measurement of the current value. Under the condition that a measuring loop is disconnected, electrons in the electrochemical reaction stay on the surface of a working electrode, the electrons are continuously enriched along with the continuous progress of the electrochemical reaction, the chemical potential on the right side of a reaction equation shown in fig. 2 is continuously increased and gradually equal to the chemical potential on the left side of the reaction equation, and the reaction equilibrium state is reached, and in the unit reactions shown in fig. 2, the oxidation state and the reduction state of glucose oxidase are described; and hydrogen peroxide and oxygen self-circulate during the reaction; glucose, gluconic acid, and the density of electrons (i.e., the amount of electricity) enriched on the working electrode determine the chemical potential on both sides of the reaction equation. Thus, there is a correlation between glucose and electrons, and the amount of electricity can be used to reflect the glucose concentration. The electric quantity is measured as the integral of the instantaneously changing current with respect to time, i.e. when the disconnected circuit is on, although the current during discharge decays with time, the absolute value of the instantaneous current is much greater than the constant current when the circuit is in a continuous on state. Therefore, when the current signal is weaker, the electric quantity in the disconnected circuit can be intermittently recorded, read and processed in data so as to realize continuous monitoring of the blood glucose value. The glucose concentration may be obtained under known electrical conditions by an empirical formula between electrical quantity and glucose concentration.

According to the dynamic relation between the glucose concentration and the electric quantity value or the current value, the electric quantity value or the current value in the loop is measured, the blood glucose concentration is accurately calculated, the accuracy of the blood glucose concentration measured value can be better ensured, and the practicability of the microneedle patch is ensured.

As a preferred embodiment, the detection component is immobilized on the working electrode.

As a preferred embodiment, the detection component is immobilized or encapsulated in a first transdermal microneedle matrix.

The detection component in the electrochemical reaction cell may be immobilized by coating on a working electrode in the electrochemical reaction cell, or more preferably, the detection component is mixed with the matrix material of the first transdermal microneedle and integrally formed in the microneedle. The amount of immobilization in the microneedle patch varies from one assay component to another. When the detection component is immobilized on the working electrode, an alternative method is to coat the detection component on the working electrode and then embed the working electrode coated with the detection component in the first transdermal microneedle matrix. The selective physical encapsulation of the first transdermal microneedle matrix for the detection component is achieved by subsequent processing such as freeze-thaw crosslinking after microneedle cast molding or 3D printing molding.

The existing fixing modes of the detection components in the electrochemical reaction cell comprise the following steps: (1) The adsorption method achieves the aim of immobilization through the interaction of secondary bonds on the surface of the carrier and the surface of the detection, but the binding force between the detection component and the carrier is weak, and the detection component and the carrier are easy to fall off and run off; (2) The embedding method is that after the carrier is mixed with the solution of the detection component, the polymerization reaction is carried out by the initiator, the detection component is limited in the network of the carrier through the physical action, but the size of the carrier network is difficult to regulate and control, the detection component is easy to leak, the initiator needs to be solidified and crosslinked by ultraviolet light, and the enzyme is easy to be partially deactivated; (3) The crosslinking method is a method of forming a network structure by cross-linking detection molecules with each other by using a bifunctional or multifunctional reagent to carry out a crosslinking reaction between the detection molecules and the carrier, and the conventional crosslinking agent is glutaraldehyde, but this method easily causes partial inactivation of enzymes.

If the detection component of the enzyme is gradually hydrolyzed and deactivated in the body fluid, the measured value of the analyte concentration level may deviate when the content of the detection component is reduced enough to maintain the reaction rate to be first order for the analyte concentration. Therefore, the effective life of the patch can be regulated and controlled by the loading of the detection component in the microneedle when the microneedle patch is manufactured, and the loading of the detection component is regulated according to the concentration level of the analyte.

As a preferred embodiment, the detection component is immobilized in a first transdermal microneedle matrix, and the amount of immobilization of the detection component is determined based on the actual range concentration of the analyte. There may be exemplified fixed amounts such as 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, the content ratio being a ratio of the weight of the detection component to the sum of the weight of the substrate and the weight of the detection component of the first transdermal microneedle, and the content ratio may be even higher.

As a preferred embodiment, the length of the first transdermal microneedle is in the range of 600-1500 μm, for example in the range of 700-1300 μm, 750-1250 μm, 800-1200 μm, 850-1100 μm, and the values are for example 900 μm, 950 μm, 1000 μm, 1050 μm, 1150 μm, 1250 μm, etc., and the length of the first transdermal microneedle includes but is not limited to the above value ranges and points.

The diameter at the maximum cross-sectional area of the first transdermal microneedle is in the range of 200-700 μm, e.g., 250-600 μm, 300-550 μm, 350-500 μm, e.g., 400 μm, 650 μm, etc., and includes, but is not limited to, the above numerical ranges and points.

The length of the first transdermal microneedle of the present invention is in the range of 600-1500 μm, and reaches the dermis layer having a thickness of about 1-2mm after penetrating the epidermis layer having a thickness of about 75-150 μm, and the penetration of the first transdermal microneedle into the epidermis does not cause pain substantially because the length of the first transdermal microneedle does not contact or less contact the nerve. Through the design to first transdermal microneedle length and diameter, first transdermal microneedle also belongs to reversible deformation to the epidermis, and the skin after removing can resume the original state after several hours, need not to insert skin with the help of having 5-7mm rigidity pointed end messenger electrochemical reaction pond, alleviates the pain sense and the confliction emotion of awaiting measuring the patient by a wide margin.

As a preferred technical scheme, one end of the working electrode in the electrochemical reaction cell is inserted into the first transdermal microneedle matrix, and the other end extends out of the patch to form a first electric contact.

Through forming first electric contact department, make the microneedle paster when being connected with outside electrical signal acquisition equipment, the operation is more convenient, swift, simple.

As a preferred embodiment, one end of the reference electrode is inserted into the second transdermal microneedle matrix, and the other end extends out of the patch to form a second electrical contact.

Through forming the second electric contact department, make the microneedle paster when being connected with outside electrical signal acquisition equipment equally, the operation is more convenient, swift, simple.

As a preferred embodiment, the working electrode in the electrochemical reaction cell comprises at least an inert conductor embedded in a first transdermal microneedle matrix.

Inert conductors in the present invention refer to conductors that do not dissolve in the electrochemical reaction.

As a preferred technical scheme, the inert conductor is selected from one or a combination of a plurality of platinum, gold, copper, silver and carbon materials.

As a preferred embodiment, the working electrode further comprises a terminal electrically connected to the inert conductor and transmitting an electrical signal formed on the inert conductor to the outside of the first transdermal microneedle.

As a preferable technical scheme, the inert conductor is a platinum wire, the binding post is a copper rod, one end of the platinum wire is inserted into the copper rod to form a working electrode, and one end of the copper rod, which is far away from the tip of the first transdermal microneedle, extends out of the patch substrate layer to form a first electric contact position.

As a preferable technical scheme, the diameter of the platinum wire is 0.1-0.18mm, and the diameter of the copper rod is 0.8-1.4mm.

As a preferred embodiment, the reference electrode may have the same or different composition as the working electrode.

As a preferred technical scheme, the material of the reference electrode includes, but is not limited to, one or a combination of several of Ag/AgCl, platinum, gold, copper, silver and carbon materials.

Because the volume of the microneedle is smaller, the working electrode is smaller, the operation is more complicated when the working electrode is connected with external detection equipment, the convenience of connection of the working electrode and the external detection equipment and the stability of electric signal conduction are improved through the introduction of the binding post, and the convenience of connection of the reference electrode and the external detection equipment is also improved when the binding post is arranged on the reference electrode.

As a preferable technical scheme, the first transdermal microneedle substrate is prepared from hydrophilic polymers.

The first transdermal microneedle matrix prepared from the hydrophilic polymer material has good biocompatibility in skin, can better keep the activity of the detection component fixed in the first transdermal microneedle matrix, prolongs the survival time of the detection component in the first transdermal microneedle matrix, and realizes continuous and accurate monitoring of blood sugar by the microneedles in body fluid.

As a preferable technical scheme, the preparation raw materials of the first transdermal microneedle matrix at least comprise polyvinyl alcohol as a main material. The inventor considers that the microneedle is made of a polymer material with better biological affinity, but the fixation of the detection component can be realized by forming the polyvinyl alcohol into a gel with a net structure, the analyte can be diffused into the interior of the gel, and the polyvinyl alcohol can be dissolved in body fluid. The method of forming the polyvinyl alcohol into the network gel is classified into a chemical crosslinking method which requires adding a crosslinking agent or an initiator into the polyvinyl alcohol to form a new chemical bond under the action of ultraviolet rays to achieve crosslinking, and a physical crosslinking method which is classified into ionic crosslinking which requires charging the polyvinyl alcohol and then reacting with a polyvalent counter ion solution to form crosslinking, and a microcrystalline construction which allows the inside of the polyvinyl alcohol to form a microcrystal to achieve crosslinking. The inventors believe that the chemical crosslinking requires the addition of a crosslinking agent or initiator to the polyvinyl alcohol, but most of the crosslinking agents or initiators have high toxicity, are harmful to the human body, and ultraviolet rays have a large influence on the activity of the detection component, and the inventors cannot use the chemical crosslinking based on the above considerations. However, ionic crosslinking requires charging of polyvinyl alcohol and also occurs in multivalent counter-ionic solutions, which is not applicable in humans. The inventors have unexpectedly found that polyvinyl alcohol is capable of having a network structure that swells but does not dissolve after absorbing body fluids by a freeze-thaw process and is in a hard glassy state in a dry state with skin penetration hardness. The inventor also found that the cross-linking of the network structure achieved by the freeze-thaw process is safer for the active detection component than the cross-linking achieved by the ultraviolet curing process, the material is also safer for implantation into the human body under the process, the activity of the detection component is not affected, the effective utilization rate of the detection component is improved, the accuracy of the detection process is reliably ensured, and the practicability of the microneedle patch is ensured. The microneedle patch provided by the invention has the advantages of simple structure, high detection accuracy and long in-vivo use time, and can be used for continuously monitoring analytes, so that the microneedle patch has an industrialization prospect in the field of continuously and accurately monitoring analytes. The inventor finds that the segment of the polyvinyl alcohol can form ordered micro-areas called microcrystals or micro-crystalline domains through freeze-thawing treatment, and the microcrystals or micro-crystalline domains are not separated in a thawing state, so that a swelling and insoluble network structure can be formed after water absorption, and the hardness of the microneedle during drying is improved. The active detection component is embedded in the polyvinyl alcohol with the net structure, so that the activity of the detection component is not influenced, and the physical process of freezing and thawing does not influence the activity of the detection component, so that the detection component can maintain a natural conformation when a catalytic reaction occurs, and the effective utilization rate of the detection component is improved. By increasing the number of freeze-thaw treatments, the density of the microcrystalline domains is increased, thereby allowing the crosslink density of the first transdermal microneedle matrix to be controlled. And polyvinyl alcohol is a chemically inert pharmaceutical adjuvant, has better biocompatibility, even has been used as a material for soft contact lenses, and can reduce inflammatory reaction and rejection reaction in body tissues when the microneedles are inserted into the skin.

The freezing temperature of the invention can be selected from-5 ℃ to-25 ℃ or-10 ℃ to-20 ℃. The thawing temperature according to the invention can be regarded as being at room temperature.

As a preferred embodiment, the polyvinyl alcohol is a fully-alcoholyzed polyvinyl alcohol and/or a mixture of a fully-alcoholyzed polyvinyl alcohol and a partially-alcoholyzed polyvinyl alcohol.

The degree of alcoholysis of the PVA is 99% +/-2%, the degree of alcoholysis of the PVA is 88% +/-2%, and the degree of alcoholysis of a small amount of PVA is 78% +/-2%.

The inventor finds that the problem that the microneedle made of the completely alcoholyzed polyvinyl alcohol has excessive deformation and uncontrolled morphology during the process of dehydration, drying and shrinkage, such as too short, too thin or bending of the microneedle caused by excessive shrinkage of the microneedle, so that the operation difficulty of the electrode during the insertion of the microneedle is increased or the formed microneedle is difficult to penetrate into the skin. The inventors have unexpectedly found that the use of a mixture of partially and fully alcoholized PVA can effectively improve the deformation problem of the microneedles, and that the microneedles can better penetrate the skin, while the use of a mixture of a small amount of alcoholized and fully alcoholized PVA can improve the deformation problem of the microneedles, but can reduce the ease with which the microneedles penetrate the skin, and that the microneedles are relatively difficult to penetrate the skin. The inventor considers that possible reasons are that by adding PVA which is added with partial alcoholysis, the hydrogen bond amount in the microneedle is reduced, the problem of overlarge internal stress caused by hydrogen bond interaction in the microneedle drying process can be reduced, the deformation rate of the microneedle matrix is reduced, the microneedle keeps a better form, the too few hydrogen bonds have an influence on the crosslinking degree of the microneedle matrix, and the crosslinking density and hardness of the microneedle matrix are reduced.

The weight average molecular weight of polyvinyl alcohol commonly used in the art is in the range of 10kDa to 130 kDa.

As a preferred technical scheme, when preparing the matrix of the first transdermal microneedle, polyvinyl alcohol is prepared into a polyvinyl alcohol solution, and the concentration of the polyvinyl alcohol solution is in the range of 18-40 wt%.

The concentration of the polyvinyl alcohol solution is, for example, 20wt%, 22wt%, 25wt%, 27wt%, 29wt%, 30wt%, 32wt%, 34wt%, 36wt%, 38wt%, and the concentration of the polyvinyl alcohol includes, but is not limited to, the above numerical points.

As a preferred technical scheme, the preparation raw materials of the first transdermal microneedle substrate can further comprise auxiliary materials, wherein the auxiliary materials are selected from one or a mixture of more of dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate, sodium carboxymethyl cellulose and polyethylene glycol.

As a preferred technical solution, in preparing the matrix of the first transdermal microneedle, the adjuvant polymer is configured into an adjuvant polymer solution, and the concentration of the adjuvant polymer solution is in the range of 5wt% to 40wt%, for example, 7wt%, 9wt%, 11wt%, 13wt%, 15wt%, 17wt%, 20wt%, 22wt%, 25wt%, 27wt%, 29wt%, 30wt%, 32wt%, 34wt%, 36wt%, 38wt%, etc. The concentration of the auxiliary material polymer includes, but is not limited to, the above numerical points.

As a preferable technical scheme, when the preparation raw material of the matrix of the first transdermal microneedle is a mixture of polyvinyl alcohol and an auxiliary material macromolecule, the mass ratio between the polyvinyl alcohol solution and the auxiliary material macromolecule solution is in the range of 3/1-8/1. The mass ratio between the polyvinyl alcohol solution and the auxiliary material polymer solution is, for example, 4/1, 5/1, 6/1, 7/1 or in the range between the above values. Preferably, the mass ratio of the polyvinyl alcohol solution to the auxiliary material polymer solution is 3/1-8/1, and the transdermal microneedle has good hardness and affinity for biomolecules and is easy to cast. Besides the non-covalent crosslinking of the material of the first transdermal microneedle substrate and the water absorption swelling insolubility, the application environment of the detection component can be optimized by adding other polymers such as dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate, sodium carboxymethylcellulose and polyethylene glycol, and regulating the crosslinking density, namely the mesh size of the crosslinking structure and the swelling performance of the microneedle. These materials are compatible with PVA and are bio-molecular friendly, providing a good immobilization environment for the active detection molecules.

An apparatus for processing electrical signals generated by a microneedle patch for continuous monitoring or detection of an analyte in a body, comprising at least a signal processor electrically connected to a measurement circuit formed by a microneedle patch for continuous monitoring or detection of an analyte in a body as described above, monitoring the electrical signals in the measurement circuit and converting the electrical signals into digital signals related to the level of the analyte.

A device for continuously monitoring or detecting an analyte in a body, comprising at least a microneedle patch as described above, said microneedle patch having adhered thereto an adhesive layer for adhering to the skin; and

The quantitative relation of the electric signal and the analyte level is input into the signal processor in the technical scheme, the electric signal in the detection loop of the signal processor can be converted into a digital signal related to the analyte level, and the signal processor capable of realizing the functions can be taken as an implementation mode of the technical scheme, and an A/D converter is exemplified as the signal processor.

As a preferable technical scheme, the device for continuously detecting the in-vivo analyte further comprises a connecting plate, wherein plug-in elements are arranged on two sides of the connecting plate, the microneedle patch is detachably connected with the connecting plate through the plug-in elements on one side of the connecting plate, the signal processor is detachably connected with the connecting plate through the plug-in elements on the other side of the connecting plate, and a measuring loop formed by the signal processor and the microneedle patch is electrically connected through the connecting plate.

The plug element in the invention is an original element capable of realizing detachable insertion and detachment, and can be exemplified by elements with plug functions such as elastic clips, plug connectors and the like. The microneedle patch is detachably connected with the connecting plate, when the microneedle patch needs to be replaced, the microneedle patch is directly detached from the connecting plate and replaced by a new microneedle patch, a new device for continuously detecting in-vivo analytes is formed, and the signal processor on the connecting plate can be repeatedly used. The signal processor is detachably connected to the connecting plate, and when the signal processor is in fault and needs to be replaced, the signal processor can be directly detached and replaced by a new device without affecting the use of other devices.

The microneedle patch and the related signal reading device can be worn on the body to realize continuous monitoring or detection of the content or concentration of biological substances in the body.

Alternatively, a method of the present invention for continuously monitoring or detecting an analyte in a body comprises at least the steps of:

step 1: casting the matrix forming preparation liquid of the first transdermal micro needle and the matrix forming preparation liquid of the second transdermal micro needle into a casting mould with a sharp tail end respectively, casting the matrix forming preparation liquid of the first transdermal micro needle into micro needle holes for forming the first transdermal micro needle on the casting mould, casting the matrix forming preparation liquid of the second transdermal micro needle into micro needle holes for forming the second transdermal micro needle on the casting mould, and then continuously casting the forming preparation liquid of the micro needle patch substrate layer into the casting mould, wherein the casting mould is placed into a running sucking device, and sucking the forming preparation liquid of the micro needle matrix into the micro needle holes through the sucking device;

Step 2: freezing and thawing the matrix molding preparation liquid of the first transdermal microneedle and the matrix molding preparation liquid of the second transdermal microneedle in a casting mould;

step 3: inserting a working electrode and a reference electrode into the cast first transdermal microneedle and the cast second transdermal microneedle, wherein the tail ends of the working electrode and the reference electrode extend out of the surface of the molding preparation liquid;

step 4: repeating the step 2 for a plurality of times; and

Preparation procedure example

In describing the specific embodiment of the present invention, the electrochemical reaction when the detection component is glucose oxidase is taken as an example, specifically describing the detection principle when the first transdermal microneedle and the second transdermal microneedle form a measurement loop, and the detection principle is also applicable to the other detection components capable of undergoing electrochemical reaction.

A method of preparing a microneedle patch for continuous detection of an analyte in a body, comprising the steps of:

(1) Preparing a casting solution of a first transdermal microneedle: 4g of 20wt% aqueous PVA, 1g of 10wt% aqueous dextran and a quantity of glucose oxidase were mixed and stirred until the mixed solution was homogeneous and stable without phase separation, the PVA brand: national drug group 124, the dextran purchased from Sigma-Aldrich;

(2) Preparing a casting solution of a second transdermal microneedle: the casting solution of the second transdermal microneedle is different from the casting solution of the first transdermal microneedle in that the casting solution does not contain glucose oxidase, and the rest raw materials and the preparation method are the same;

(3) Casting solution of patch substrate layer of microneedle patch: the patch substrate layer adopts a PVA aqueous solution with the concentration of 22 weight percent, and the brand of PVA: a national drug group 124;

(4) Casting of microneedle patches: the method comprises the steps of placing a casting mould of a breathable polymer material on a running suction device, wherein the breathable polymer casting mould is provided with microneedle holes for casting the microneedles, casting a casting solution of the first transdermal microneedles and a casting solution of the second transdermal microneedles on different microneedle holes of the mould, sucking the casting solution into the microneedle holes through the suction device, stopping running the suction device when the microneedle holes are filled with casting solution, placing an auxiliary mould for preventing solution loss of a patch substrate layer on the casting mould, casting the casting solution of the patch substrate layer in the auxiliary mould, and forming a microneedle patch for continuously monitoring or detecting analytes in a body after casting of each three microneedle holes, wherein one microneedle hole is the second transdermal microneedles;

(5) Freezing-thawing of microneedle patches: freezing the casting mold with the solution cast and the auxiliary mold for preventing the solution from losing in a refrigerator at the temperature of minus 20 ℃ for 30 hours, and thawing for 30 minutes at room temperature;

(6) Insertion of the electrode: inserting a platinum wire into the center of a first transdermal microneedle, inserting a platinum wire into a reference electrode in the copper bar into the center of a second transdermal microneedle, wherein the platinum wire has a diameter of 0.15mm and a length of 1.1mm, wherein the length in the microneedle is 0.7mm, the length in the copper bar is 0.4mm, the diameter of the copper bar is 1.2mm, and the length of the copper bar needs to extend out of a patch substrate layer;

(7) Re-freeze-thaw treatment: freezing the microneedle communication mould inserted with the electrode in a refrigerator at-20 ℃ for 21h again, taking out, thawing for 3.5h at room temperature, and repeating the thawing for 3 times;

(8) Drying of microneedle patches: and after repeated freezing-thawing, taking out the micro-needle patch from the refrigerator, placing the micro-needle patch at room temperature, naturally drying and shrinking the micro-needle patch for 24 hours, tearing off the micro-needle patch in a mould, clamping the micro-needle patch by a clamp, and placing the micro-needle patch in a dryer for drying until the micro-needle patch is completely dried. Wherein the dried lengths of the cast first and second transdermal microneedles are in the range of 900 μm and the diameter at the maximum cross-sectional area is in the range of 300 μm.

The glucose solutions of different concentrations were prepared by dissolving the desired amount of glucose in PBS solution at pH 7.0, available from ThermoFisher, and glucose from Sigma.

The microneedle patches used in the following examples were all prepared based on the above preparation examples, and specifically adjusted according to the required content of glucose oxidase, wherein the proportion of glucose oxidase is as follows: the weight of glucose oxidase is the ratio of the weight of the first transdermal microneedle matrix to the sum of the weight of glucose oxidase.

The definition of blood glucose level is generally divided into three cases, namely, hyperglycemia, namely, the concentration of glucose is more than 7mmol/L, normal blood glucose, namely, the concentration of glucose is 3.9-7mmol/L, and hypoglycemia, namely, the concentration of glucose is less than 3.9mmol/L. When the scheme is used for experiments, the range of the glucose concentration is 1-36mmol/L, and the change range of the glucose concentration in actual human body tests can be met.

Example 1

For verifying the detection and monitoring effect of the microneedle patch in the technical scheme, a detection device shown in fig. 1 is constructed, wherein the detection device comprises two first transdermal microneedles 1, one second transdermal microneedle 6 and working electrodes inserted into the first transdermal microneedles 1, each working electrode is formed by inserting a platinum wire 2 into a copper rod 3, the copper rod 3 extends out of a patch substrate layer 4 to form a first electric contact part, a groove 12 is formed in the side wall of the copper rod 3, the copper rod is clamped in the patch 4 through the groove 12, and the bonding strength between the copper rod 12 and the patch 4 is improved. The first transdermal microneedle 1 contains glucose oxidase, the second transdermal microneedle 6 is inserted with a reference electrode, the reference electrode is also formed by inserting a platinum wire into a copper bar, the copper bar extends out of the patch 4 to form a second electric contact, the two parallel first electric contacts and the second electric contacts are electrically connected to form a measuring loop, the nanoampere meter 5 is electrically connected in the loop, the current in the loop is measured, and the nanoampere meter can select a Keysight digital multimeter. In operation, the microneedle patches in the test device were inserted into a 0.5mm thick dermis layer of a polyvinyl alcohol electrospun fiber simulation placed on a transdermal cell loaded with solutions of different glucose concentrations. The polyvinyl alcohol electrospun fibers contact and absorb the glucose solution, allowing the first and second transdermal microneedles inserted therein to swell. The glucose in the solution rapidly diffuses into the first transdermal microneedle and the second transdermal microneedle which are swelled into a hydrogel state, and the glucose entering the first transdermal microneedle is catalyzed and oxidized by glucose oxidase carried in the glucose to generate gluconolactone and then is hydrolyzed into gluconic acid; the catalytic site of the glucose oxidase is converted into a reduced state, and the glucose oxidase in the reduced state is oxidized into an oxidized state with catalytic activity under the action of oxygen in body fluid, and simultaneously one molecule of hydrogen peroxide is released; the hydrogen peroxide loses two electrons on the surface of the platinum wire of the working electrode to generate oxygen and two hydrogen ions of a molecule, the reaction principle is shown in figure 2, and the electrons are detected by the nanoampere meter 5 in the process of transmitting the electrons to the reference electrode by the working electrode to form a readable current value.

Example 2

Under the condition of ensuring sufficient glucose oxidase carrying capacity, examining the change value of current with time after a measuring loop is connected, preparing a microneedle patch with 10wt% of glucose oxidase content in a first transdermal microneedle, preparing a glucose solution with the concentration of 12mM, connecting the measuring loop after reacting for 4min at 37 ℃, and measuring the change curve of current with time through the microneedle patch to obtain a current-time curve shown in figure 3. As can be seen from fig. 3, when the measuring circuit is not turned on, electrons in the electrochemical reaction are not removed from the surface of the working electrode, a chemical potential driving the reverse reaction is formed, electrons concentrated on the surface of the working electrode are rapidly moved to the reference electrode at the moment of turning on the measuring circuit, a high instantaneous current 158nA is formed, the current continuously decays to 35nA with time and is constant at the value after 3s, the constant current value can reflect the glucose concentration, the constant current value at any time point after 3s can be selected as the current value of the reaction glucose concentration during the detection, the current value after 8s of the measuring circuit is selected as the constant current value in the subsequent experiment, the test principle in the invention can be verified through the experiment, and the glucose concentration can be reflected by the constant current in the measuring circuit when the electric signal is strong.

Example 3

In order to examine the influence of the glucose oxidase loading in the first transdermal microneedle on the accuracy of monitoring the blood glucose level, a microneedle patch with 3wt% of glucose oxidase content in the first transdermal microneedle, a microneedle patch with 5wt% of glucose oxidase content in the first transdermal microneedle, and a microneedle patch with 10wt% of glucose oxidase content in the first transdermal microneedle were prepared respectively, a plurality of glucose solutions with different concentrations of 1mM to 36mM of glucose concentration were prepared, and the microneedle patches with different glucose oxidase loading amounts were respectively placed in the glucose solutions with different concentrations at 37 ℃ to react, so that the microneedle can achieve a better swelling effect in the solution for 4min, after the reaction, the measurement circuit was turned on, the steady-state current value when the current was turned on for 8s was taken as a constant reaction current value, and the curve in FIG. 4 was obtained by plotting a constant reaction current-glucose concentration curve. As can be seen from fig. 4, the current no longer responds linearly to an increase in glucose concentration at higher glucose concentrations after a decrease in the glucose oxidase loading. This result supports our design idea that when the catalytic site density of glucose oxidase does not significantly exceed the glucose concentration, the electrochemical reaction rate is no longer first order for the glucose concentration. The microneedle patch with 10wt% glucose oxidase content in the first transdermal microneedle has good linear relation between the constant current value and the grape concentration of 1mM to 36mM at 37 ℃, and the test result is subjected to linear fitting to obtain a straight line after fitting as shown in fig. 5, wherein r2= 0.9968 has good linear relation, and the fitting equation is: y=2.467x+5.6154, and the fitting equation is deformed to obtain the relationship between the glucose concentration and the constant reaction current, i.e., cglu= (I-5.6154)/2.467, where CGlu is the glucose concentration in mol/L, I is the constant reaction current value in nA. The glucose concentration value can be calculated using this empirical formula under the condition of a known constant current. The microneedle patch with 10wt% glucose oxidase still has a good linear relationship between current and glucose concentration in a large glucose concentration, and in the subsequent experiments, if no special statement is made, the microneedle patch with 10wt% glucose oxidase is adopted for the experiments.

Example 4

The products of the companies such as yaban and the like are attached once, and a 6mm sensor needle is placed under the skin for 14 days; the method can be attached for 14 days, and data can be intermittently read. But shortening the attaching time is more convenient for patients, such as coping with summer heat, sweating, flushing, etc. Because the microneedle patch in this technical scheme has lower use cost, when the patient carries out activities such as cooling, removable microneedle patch. To investigate the time for accurate monitoring of glucose concentration by a microneedle patch carrying 10wt% glucose oxidase. Placing the microneedle patch on the transdermal cell continuously at 37deg.C for 14 days; the responses of the current to the glucose concentration were measured on days 1, 2, 5, 8 and 14, and the current value at 8s after the circuit was turned on was taken as a constant current value, and a curve of the relationship between the current and the glucose concentration was plotted on different days, to obtain fig. 6. As can be seen in fig. 6, the constant current and glucose concentration maintained a precise linear relationship for 1-8 days when the microneedle patch was continuously placed in a transdermal cell at 37 c, and the measured value was somewhat lower when it reached 14 days, but a substantially linear relationship was maintained. This test result shows that under 37 ℃ conditions, glucose oxidase progressively degrades and becomes inactive with increasing incubation time, such that its effective load is lower than that required to achieve a linear current response. Since the current measurement on day 14 remains linear with respect to glucose concentration, it is expected that accurate monitoring of glucose concentration for two weeks can be achieved by appropriately increasing the glucose oxidase loading. The evaporation of the glucose solution decreases as the experiment proceeds, and glucose solution of a corresponding concentration is replenished in real time during the experiment.

Example 5

Glucose solutions were prepared at concentrations of 3mM, 6mM, 9mM, 12mM, 18mM and 24mM, respectively, in the most common ranges of 3mM to 24mM for diabetics, and the prepared glucose solutions were then placed in a thermostatic water bath at 37℃to obtain glucose solutions at 37 ℃. The transdermal drug delivery cells of the in vitro experiments were placed in a 37℃water bath, and then glucose solutions of different concentrations at 37℃were added to the different transdermal drug delivery cells, respectively. Glucose concentration was measured using a 10wt% glucose oxidase loaded microneedle patch, a constant current value was measured, and the measured value was substituted into the relationship cglu= (I-5.6154)/2.467 of the obtained constant current and glucose concentration to calculate the glucose concentration.

The same glucose concentration test in vitro experiment was performed using a commercially available yaban continuous monitor glucometer Abbott Free Style Libre 3. The transdermal drug delivery cells were placed in a constant temperature shallow water bath at 37℃and then glucose solutions at 37℃were added to them at 3-24mM, respectively. The glucose sensor is mounted on a disposable ejection device according to the description on the continuous monitoring glucometer of the yaban, and the ejection device is pressed, so that the sensor probe instantaneously pierces the 0.5mm thick simulated dermal tissue PVA fibrous membrane. And (3) placing a sensor probe with a PVA fiber membrane on a transdermal drug delivery tank to react with glucose solution, continuously monitoring the glucose concentration by using a scanning detector scanning sensor of a self-contained scanning glucose meter in a yaban manner, and recording the glucose concentration after the value is stable. The sensor with the PVA fiber film is taken out and placed in a glucose solution with the next concentration, and the measured glucose concentration value is recorded.

The graph line drawn between the glucose concentration value and the real glucose concentration value of the microneedle patch test, the graph line drawn between the glucose concentration value and the real glucose concentration value of the commercial yapei continuous monitoring glucometer test, and the graph line drawn by the real glucose concentration value are used as comparison graph lines to obtain the graph figure 7. As can be seen from fig. 7, the glucose concentration value obtained by using the microneedle patch is closer to the glucose concentration value actually added, and the microneedle patch of the present invention has higher detection accuracy.

Example 6

To further investigate the relationship of the amount of charge enriched on the first percutaneous microneedle working electrode to the measurement loop off time. Under the condition of constant room temperature at 25 ℃, a microneedle patch with 10wt% glucose oxidase loading is adopted to react with 12mM glucose solution, the circuit breaking time is changed, the current-time curve under different breaking times is measured, the area under the curve is integrated to obtain the enriched electric quantity value on the working electrode, and the relation curve between the enriched electric quantity value on the working electrode and the circuit breaking time is drawn to obtain the graph of FIG. 8. As can be seen from fig. 8, as the off time of the measurement circuit increases, the value of the enriched electric quantity on the working electrode increases, and finally reaches an equilibrium value, and the value of the enriched electric quantity on the working electrode reaches an equilibrium value at about 4min under the reaction condition in this embodiment.

Example 7

And respectively adopting a microneedle patch with the glucose oxidase content of 3wt% in the first transdermal microneedle, a microneedle patch with the glucose oxidase content of 5wt% in the first transdermal microneedle and a microneedle patch with the glucose oxidase content of 10wt% in the first transdermal microneedle, preparing glucose solution with the glucose concentration of 1mM to 36mM, respectively placing the microneedle patches with different glucose oxidase loading amounts in the glucose solution with different concentrations at 37 ℃ for reaction, integrating the area under the curve in a current release curve 2s by a communication measuring loop to obtain an enrichment electric quantity value on a working electrode, changing the circuit breaking time of the circuit, measuring the enrichment electric quantity on the electrode under different breaking times until the enrichment electric quantity on the electrode is saturated, and taking a relation curve of the saturation electric quantity value and the glucose concentration to obtain a graph 9. As can be seen from fig. 9, the value of the accumulated saturated electric power on the working electrode does not significantly change when the reaction reaches equilibrium with a decrease in the enzyme loading. In experiments we found that the change in enzyme load affects the time to saturation of the enriched charge on the working electrode where the reaction reaches equilibrium after one charge measurement. When the glucose oxidase loading in the first transdermal microneedle was 10wt%, the time to saturation of the charge on the working electrode was 4min. When the glucose oxidase loading in the first transdermal microneedle was reduced to 5wt% and 3wt%, the on-electrode charge enrichment saturation time was 7min and 11min. It is seen from this that how much the glucose oxidase loading affects the time for the chemical reaction to reach equilibrium.

Example 8

To further examine the effect of glucose concentration on the time to saturation of charge enrichment on the working electrode, the concentration of glucose was tested at 6mM, 12mM, 24mM and 36mM, respectively, for the concentration of glucose on the microneedle patch working electrode versus circuit break time, and the working electrode saturation enrichment at different glucose concentrations versus break time was plotted to give the graph of FIG. 10. It can be seen from fig. 10 that as the glucose concentration increases, the saturation electric power value on the working electrode also increases.

Example 9

The in vitro test in example 8 was performed at 25 c, since the human body temperature was around 37 c, and this example further examined the effect of temperature on the value of the saturation electric energy enriched on the working electrode. The values of the enrichment saturation electric quantity and the time of the electric quantity enrichment saturation on the working electrode were measured by reacting the microneedle patches with 10wt% glucose oxidase loading with 3mM, 6mM, 12mM and 24mM glucose solutions at 25℃and 37℃respectively, and the relationship curves between the enrichment saturation electric quantity and the glucose concentration at different temperatures were plotted to obtain FIG. 11. From fig. 11, it can be seen that there is no significant difference in the value of the enriched saturated electric power on the working electrode measured at different temperatures (P > 0.05).

Example 10

As is clear from example 2, the current tends to decay within 2s after the open circuit is turned on, and the current reaches a steady state after 2 s. To investigate the relationship between glucose concentration and the value of the saturation electric charge enriched on the working electrode of the microneedle patch when the reaction reached equilibrium. Reacting a microneedle patch with 10wt% glucose oxidase load with glucose solutions with different concentrations and at 37 ℃ for more than 4min, enriching charges on a working electrode to reach saturation, connecting a circuit, measuring a current attenuation curve, integrating the area under the curve within 2 seconds to obtain an enriched saturated electric quantity value on the working electrode, drawing a relation curve of the saturated electric quantity value and the glucose concentration to obtain a graph 12, and performing linear fitting on the graph 12 to obtain a curve shown in the graph 13, wherein R in the graph 13 is shown as a curve ² = 0.9804, with a good linear relationship, by obtaining the fitted curve in fig. 13, an empirical formula is obtained, through which the corresponding glucose concentration value can be calculated under the condition of knowing the saturated electric quantity of the working electrode.

Example 11

The enzyme can be deactivated during the use of the electrochemical reaction cell microneedle due to factors such as the environment. In this example, an in vitro experiment of microneedle glucose concentration in an electrochemical reaction cell was performed for 14 consecutive days in a glucose solution at 37℃using an coulometry. The 10wt% glucose oxidase loaded microneedle patches were reacted with 3mM, 9mM, 18mM and 30mM glucose solutions, respectively, and the microneedle patches were placed successively on transdermal cells of different glucose concentrations for 14 days, and the enrichment saturation electric power values on the working electrodes were tested at day 1, day 2, day 5, day 8 and day 14, respectively. And drawing a relation graph between the saturated electric quantity value enriched on the working electrode and the glucose concentration on different days to obtain a graph 14. As can be seen from fig. 14, the change in the value of the enriched saturated electrical quantity on the electrode measured over the different measurement days was not significant (P > 0.05) over the 14 days. From this test, it can be seen that the microneedle patch can be used to continuously monitor glucose concentration changes by monitoring the electrical value. We examined the time for the charge enrichment to saturate on the platinum wire working electrode after one charge measurement at the same time, the time for the charge enrichment to saturate on the electrode was 4 minutes in the first 8 days, and the time for the charge enrichment to saturate was prolonged to 5 minutes on the 14 th day. For enzyme degradation in the use process of the micro-needle of the electrochemical reaction tank, the intermittent electric quantity method can be adopted to solve the problem by prolonging the electric quantity enrichment time. The evaporation of the glucose solution decreases as the experiment proceeds, and glucose solutions of corresponding concentrations are replenished in real time during the experiment.

Example 12

The microneedle patch with 6.1wt% glucose oxidase loading was applied to a living rabbit for in vivo testing, and the specific implementation process and experimental results are as follows:

1. reagent and related instrument

Glucose was purchased from: sigma; multimeter and software: keyweight; the method comprises the steps of carrying out a first treatment on the surface of the Blood glucose meter: rogowski, luo Kangquan active; test paper: rogowski; medical sterilized cotton ball: european cleaning; blood taking needle: rogowski; depilatory cream: weiting.

2. Animal experiment

(1) Animals:

adult New Zealand white rabbits were purchased from Shanghai Jia Gao Biotech Co. Production license number: SCXK 2010-0028 (Shanghai). Weight of: 2-2.5kg. The number is 6. Grade: a normal stage. Gender: and (3) female. Animal feeding and experiments are carried out in special animal houses of experimental animal centers of Shanghai university, each single cage is fed, the experiment is started after one week of adaptive feeding of rabbits, and common rabbit feed is fed normally.

(2) The animal experiment method comprises the following steps:

after the adaptive feeding, the following experiments were performed:

1. the rabbit monaural dehairing paste is used for dehairing, a microneedle patch is attached to the dehaired rabbit ear, an application film with a side adhesive layer is attached to the microneedle patch, at least the electric contact part is ensured to be exposed out of the application film, the microneedle patch is firmly attached to a monitored body through the application of the application film, and fig. 15 is an exemplary embodiment, wherein the application film 9 is attached to the microneedle patch substrate layer 7, and the first electric contact part 8 and the second electric contact part are leaked so as to be electrically connected with an external device. When in use, 3 micro-needles are forcefully inserted into the skin of the single ear of the rabbit by the thumb pressing the middle part of the micro-needle patch, and then the adhesive film is removed to firmly adhere the micro-needle patch to the ear of the rabbit;

2. Rabbits were fed in single cages with a fasting period of 12 hours (no water forbidden for fasting) before the experiment;

3. the first day: measuring fasting blood glucose;

after the blood glucose monitoring microneedle patch reacts with glucose in body fluid for 4 minutes, a keysight software start button is opened, a universal meter starts to collect data, meanwhile, the anode and the cathode of a wiring pen on the instrument are respectively communicated with a reference electrode and a working electrode on the microneedle patch again, 40 seconds is measured, and the data is exported by pressing a stop button; .

4. Meanwhile, blood glucose measurement is carried out on the opposite side ear of the ear to which the microneedle patch is attached by adopting an auricular vein blood taking Rogowski blood glucose meter, and the blood glucose measurement method is carried out by using the Rogowski blood glucose meter: sterilizing rabbit ears with 75% alcohol cotton, puncturing skin with special blood taking needle to squeeze bleeding, sucking trace blood with front end of Rogowski professional blood glucose test paper, and inserting into blood glucose meter to read blood glucose value; .

5. Intravenous injection of an amount of a 50wt% strength glucose solution into the rabbit ear margin; .

6. Measuring blood glucose values 15min, 30min, 1h, 2h, 3h and 4h after glucose injection is completed; .

After the measurement is finished, the rabbits are fed with feed normally; .

7. When a plurality of days of testing is needed, the steps 2-6 are repeated every day in the following steps.

Selection of electrical signals for microneedle patch testing of New Zealand white rabbits

One of six New Zealand white rabbits was selected, and after 12 hours of fasting, a microneedle patch was attached to a single ear of the rabbit, and blood glucose level was measured after 4 minutes, and a curve of current versus time was obtained by test software, as shown in FIG. 16. From fig. 16, it can be seen that the blood glucose of the rabbit was measured using the microneedle patch, and the measured current value was rapidly decreased and then reached a constant current within 2 seconds, and the constant current was small, which was 0.0X nA. The glucose content in tissue fluid is measured in rabbit experiments, the tissue fluid quantity in animals is obviously reduced compared with the external glucose solution quantity, and the microneedle swelling and glucose diffusion speed are reduced, so that the reaction between glucose oxidase and glucose is slowed down. From fig. 16, it can be seen that the constant current value detected by the microneedle patch when measuring the glucose concentration in the tissue fluid of the rabbit is very small, so that in the subsequent experiment, we obtain the electric value by integrating the instantaneous current time, and calculate the glucose concentration by combining the chemical kinetics relation between the electric value and the glucose concentration with the empirical formula in example 10.

New Zealand white rabbits screening of glucose injection dosage

Optionally, a rabbit was fasted for 12 hours, and then blood glucose levels were measured by injecting 3g/kg, 2g/kg, 1.25g/kg of 50wt% glucose solution, respectively, into the ear vein at the rabbit weight, using a rogowski glucometer at 15, 30, 60, 120, 180, 240min, respectively, and the change in blood glucose in different amounts of glucose injected is shown in table 1 below, and the graph of table 1 was plotted to obtain fig. 17. As can be seen from Table 1 and FIG. 17, when the injection amount was 3g/kg, the 15min rabbit blood glucose level exceeded the upper limit of detection of the blood glucose meter, and thus there was no reading. To more closely fit the normal blood glucose range of the human body, we selected a 50wt% glucose injection of 1.25g/kg as the experimental condition for the following animal experiments.

TABLE 1

Blood glucose monitoring results of the microneedle patch and the rogowski blood glucose meter were respectively injected into the ear veins of six rabbits at an amount of 1.25g/kg of 50wt% glucose, blood glucose values of the microneedle patch and the rogowski blood glucose meter were tested according to an animal experiment method, wherein an instantaneous current varied when a circuit obtained through the microneedle patch test was turned on, blood glucose average values of six rabbits respectively measured with the microneedle patch and the rogowski blood glucose meter were calculated through an empirical formula between the electric quantity and the glucose concentration in example 10, and a blood glucose-time curve was drawn, to obtain fig. 18. As can be seen from fig. 18, the blood glucose level measured by the microneedle patch has the same trend as the blood glucose level measured by the commercially available roche blood glucose meter, and a small range of differences exists. The blood glucose level increased rapidly after intravenous glucose injection at the rabbit ear margin, and recovered to normal blood glucose level at 2 hours. When blood glucose rises rapidly, the blood glucose level measured by the microneedle patch is slightly lower than that of blood collection, probably because blood collection blood glucose meters collect blood from capillaries, and the microneedle patch monitors that the glucose concentration in body fluid reacts later than blood glucose.

Continuous 3-day rabbit blood glucose monitoring of microneedle patch and Roche blood sampling blood glucose meter

To further compare the blood glucose values measured by the electrochemical blood glucose monitoring microneedle patch with the blood glucose meter, the study injected 50wt% glucose (1.25 g/kg) 12h on a fasting basis in 6 healthy rabbits, and the measured blood glucose values were subjected to significant differential detection. Blood glucose was monitored by microneedle patch and roche blood glucose meter for 3 consecutive days, and the average blood glucose values obtained by the test were calculated, respectively, and the test data are shown in fig. 19. Experiments were continuously performed for 3 days, and blood glucose values measured for 3 days were subjected to significant difference detection. From the graph, the blood glucose monitoring results of rabbits for 3 consecutive days show that the microneedle patch has good correlation with blood glucose values measured by a commercially available rogowski blood glucose meter. The blood sugar monitoring microneedle patch is proved to be capable of continuously monitoring blood sugar, and the result is stable and reliable.

Based on the principle of mathematical statistics, the variances of blood glucose values measured by the electrochemical blood glucose monitoring microneedle patch and the blood glucose meter are compared by using a double-sample variance F-test. If the p value is less than 0.05, the variance of the blood glucose values measured between the two groups is obviously different, and the two-sample heteroscedastic t test is needed to be used for testing; if the p value is greater than 0.05, it is indicated that there is no significant difference in variance of blood glucose values measured between the two groups, and the test is performed using a double sample equal variance t test. For further t-tests, if p > 0.05 for the resulting double-sided test, it is indicated that there is no significant difference between the two sets of blood glucose values compared to each other; if 0.01< p <0.05, then the blood glucose values measured between the two groups are obviously different; if p < 0.01, it is indicated that there is a very significant difference in the measured blood glucose values between the two groups. Wherein table 2 shows the results of the blood glucose and t-test measured by the microneedle patch and the blood glucose meter after the first day of the fasting glucose injection, table 3 shows the results of the blood glucose and t-test measured by the microneedle patch and the blood glucose meter after the second day of the fasting glucose injection, and table 4 shows the results of the blood glucose and t-test measured by the microneedle patch and the blood glucose meter after the third day of the fasting glucose injection.

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

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Glucose was intravenously injected into the ear margin of a rabbit having a hollow 12h to measure its blood glucose change, and the measurement was continued for 3 days. From the statistics in tables 2, 3, and 4, it can be seen that there is no significant difference between the microneedle patch and the blood glucose measurement of the blood glucose meter. Therefore, our blood glucose monitoring microneedle patch can be used for continuous monitoring of living blood glucose.

One rabbit is selected at will, the blood sugar monitoring microneedle patch is firmly attached to a single ear of the rabbit, blood sugar values are measured after 12h of fasting every day respectively, and meanwhile, a Roche hemostix is used for blood sampling and blood sugar measurement as a control group. The results of continuously monitoring the fasting blood glucose of the rabbits every day show that the blood glucose monitoring microneedle patch can continuously monitor the blood glucose for 14 days and the measured blood glucose value is stable. The blood glucose level measured by the blood glucose monitoring microneedle patch after 15 days was significantly lower than that measured by the blood glucose meter, probably because the glucose oxidase in the blood glucose monitoring microneedle patch was subjected to long-time electrode action and the influence of the physiological environment in the body, and the activity of part of the oxidase was ineffective. From this, it can be seen that the microneedle patch for monitoring or detecting an in-vivo analyte according to the present invention is capable of stably, chronically existing and accurately measuring a relevant signal in a living body.

While the disclosure has been provided in terms of the illustrated implementations, those of ordinary skill in the art will readily recognize that there could be variations to the embodiments and those variations would be within the scope of the disclosure. Accordingly, many modifications may be made by one of ordinary skill in the art without departing from the scope of the appended claims.

Claims

1. A microneedle patch for continuous monitoring or detection of an analyte in a body, comprising at least one first transdermal microneedle standing on a skin-contacting surface of a patch substrate layer, the first transdermal microneedle having a sharp shape at an end thereof remote from the patch substrate layer;

the electrochemical reaction cell is integrated in the first transdermal microneedle, the analyte and the detection component contained in the matrix of the first transdermal microneedle are subjected to electrochemical reaction in the electrochemical reaction cell to form an electric signal, the in-vivo concentration level of the analyte is reflected by the magnitude of the detected electric signal, the preparation raw material of the matrix of the first transdermal microneedle is hydrophilic polymer, the preparation raw material of the hydrophilic polymer at least comprises main material polyvinyl alcohol, the matrix of the first transdermal microneedle is provided with a network structure which swells but does not dissolve after water absorption formed by the polyvinyl alcohol subjected to freezing thawing treatment, the network structure allows the analyte to diffuse into or move away from the matrix of the first transdermal microneedle, and the detection component is prevented from moving away, the microneedle patch further comprises at least one second transdermal microneedle which is vertical to the surface of the patch substrate layer, the second transdermal microneedle does not contain the detection component, the reference electrode is included in the second transdermal microneedle, the electrochemical reaction cell in the first transdermal microneedle comprises a working electrode, and the second transdermal microneedle is connected with the reference electrode in the working circuit.

2. The microneedle patch of claim 1, wherein the electrical signal generated in the measurement loop comprises an electrical value in quantitative relationship to chemical equilibrium and an electrical value in quantitative relationship to electrochemical reaction rate.

3. The microneedle patch for continuous monitoring or detection of an analyte in vivo according to claim 1, wherein the length of the first transdermal microneedle is in the range of 600-1500 μm and the diameter at the maximum cross-sectional area is in the range of 200-700 μm.

4. The microneedle patch for continuous monitoring or detection of analytes in a body of claim 1, wherein the working electrode in the electrochemical reaction cell has one end inserted into the first transdermal microneedle matrix and the other end extending beyond the back of the skin facing surface of the patch substrate layer to form a first electrical contact.

5. The microneedle patch for continuous monitoring or detection of analytes in a body of claim 4, wherein one end of the reference electrode is inserted into the second transdermal microneedle matrix and the other end extends beyond the back of the skin facing surface of the patch substrate layer to form a second electrical contact.

6. The microneedle patch for continuous monitoring or detection of analytes in a body of claim 1, wherein the working electrode in the electrochemical reaction cell comprises at least an inert conductor embedded in a first transdermal microneedle matrix, the inert conductor being selected from one or a combination of several of platinum, gold, copper, silver, carbon materials.

7. The microneedle patch for continuous monitoring or detection of an analyte in a body of claim 6, wherein the working electrode further comprises a terminal post electrically connected to the inert conductor and transmitting an electrical signal formed on the inert conductor to the exterior of the first transdermal microneedle.

8. The microneedle patch for continuous monitoring or detecting in vivo analytes according to claim 7, wherein the hydrophilic polymer is prepared from an auxiliary material selected from one or a mixture of several of dextran, chitosan, alginate, hyaluronic acid, sodium hyaluronate, sodium carboxymethyl cellulose, and polyethylene glycol.

9. A device for continuous monitoring or detection of analytes in vivo, comprising at least a microneedle patch according to any one of claims 2-8, said microneedle patch having an adhesive layer adhered thereto for adhering to skin; and

and a signal processor electrically connected to the measurement loop between the working electrode of the first transdermal microneedle and the reference electrode of the second transdermal microneedle, monitoring the electrical signal in the measurement loop and converting the electrical signal into a digital signal related to the analyte level.

10. The device for continuous monitoring or detection of analytes in a body according to claim 9, further comprising a connection plate, wherein the connection plate is provided with plug elements on both sides, wherein the microneedle patch is detachably connected to the connection plate via the plug elements on one side of the connection plate, wherein the signal processor is detachably connected to the connection plate via the plug elements on the other side of the connection plate, and wherein the signal processor and the measurement circuit are electrically connected via the connection plate.

11. The device for continuous monitoring or detection of an analyte in a body according to claim 9 or 10, wherein the device is a wearable device.

12. A method for preparing a microneedle patch for continuous monitoring or detection of an analyte in vivo according to any one of claims 1 to 8, comprising the steps of:

step 1: casting the matrix forming preparation liquid of the first transdermal micro needle into the micro needle hole for the first transdermal micro needle forming on a casting mould, casting the matrix forming preparation liquid of the second transdermal micro needle into the micro needle hole for the second transdermal micro needle forming on the casting mould, placing the casting mould into a running sucking device, sucking the matrix forming preparation liquid of the first transdermal micro needle into the micro needle hole for the first transdermal micro needle forming through the sucking device, sucking the matrix forming preparation liquid of the second transdermal micro needle into the micro needle hole for the second transdermal micro needle forming through the sucking device, stopping running the sucking device when the corresponding micro needle hole is filled with the matrix forming preparation liquid of the first transdermal micro needle and the matrix forming preparation liquid of the second transdermal micro needle, placing an auxiliary mould for preventing the forming preparation liquid of the micro needle patch substrate layer on the casting mould, and casting the forming preparation liquid of the micro needle patch substrate layer on the auxiliary mould;

step 3: inserting a working electrode into the cast first transdermal microneedle, inserting a reference electrode into the cast second transdermal microneedle, and extending the tail end of the working electrode and the tail end of the reference electrode out of the surface of the microneedle patch substrate layer; step 4: repeating the step 2 for a plurality of times; and

step 5: and (3) after the casting mould is placed at room temperature and naturally dried and contracted, the microneedle patch is removed from the casting mould, clamped by a clamp and placed in a dryer for drying until the microneedle patch is dried completely.